Rigid-rod β-barrel pores as enzyme sensors

Ce travail présente le design et la synthèse d'une baguette formée d'un octiphényle portant sur chaque phényle: le pentapeptide -Leu-Arg-Leu-His-Leu. L'auto-assemblage de cette baguette forme une barrique β qui agit comme un pore dans les doubles couches lipidiques, perméable au passage d'anions. Les études de blocage avec l'acide poly-glutamique ont mis en évidence la formation d'un complexe entre le polymère et la barrique transmembranaire ainsi qu'une coopérativité dipôle-potentiel. La mise au point et l'utilisation de la structure supramoléculaire comme senseur d'enzymes ont été largement illustrées sur des biopolymères ou des enzymes intervenant dans différents processus biologiques. L'utilisation d'un seul pore permet la détection d'une large variété d'enzymes et de substrats à intérêt biologiques, sans requérir aucune modification chimique des composés impliqués. Des applications telles que la recherche d'inhibiteurs d'enzymes ou la quantification du sucre dans les boissons ont découlé de cette recherche

α-Helix Recognition by Rigid-Rod β-Barrel Ion Channels with Internal Arginine-Histidine Dyads in Polarized Bilayer Membranes

The objective of this study was to exploit multifunctional rigid-rod ß-barrel ion channels for a-helix recognition by dipole-potential interactions in polarized membranes. Synthesis and evaluation of artificial ß-barrel 1 characterized by p-octiphenyl ‘staves,' leucine residues at the outer and histidine as well as, for the first time, arginine residues at the inner barrel surface are described. Internal arginines were introduced to recognize organic ions such as a-helical poly-Image -glutamic acid (a-PLGA) passing through pores formed by barrel 1 at nanomolar concentrations. In unpolarized spherical bilayers (EYPC-LUVs), P-helical a-PLGA blocked pore 1 with a KD=150 nM at pH=4.5. As expected for a highly symmetric supramolecular host, a KD=100 nM determined for M-helical a-PDGA at pH=4.5 did not support substantial chiral guest recognition. Decreasing KD's with increasing pH indicated that, in unpolarized bilayers, pore 1 recognizes anions (rather than a-helices). In polarized spherical membranes, the KD for a-PLGA at pH=4.5 dropped substantially to 13 nM at V ˜-150 mV. This experimental support for operational dipole-potential interactions indicates that a-PLGAs bind within active pores in transmembrane orientation with intravesicular N- and extravesicular C-termini. Independence on bilayer polarization for binding of random-coil PLGA at pH=5.5 corroborated that dipole–potential interactions account for a-helix recognition in polarized membranes

On selectivity and sensitivity of synthetic multifunctional pores as enzyme sensors: Discrimination between ATP and ADP and comparison with biological pores

This report delineates scope and limitation of the selectivity of synthetic multifunctional pores as enzyme sensors using glycolytic enzymes as example (G. Das, P. Talukdar, and S. Matile, Science, 2002, Vol. 298, pp. 1600-1602). Unproblematic detectability of hexokinase and phosphofructokinase demonstrates that the selectivity of synthetic multifunctional pore (SMPs) sensors suffices to sense ATP in mixed analytes containing ADP, whereas detection of the isomerization of glucose 6-phosphate into fructose 6-phosphate by phosphoglucose isomerase is not possible with confidence. The sensitivity of SMP sensors is sufficient for end-point detection of one picomole poly-L-glutamate hydrolyzed by papain in unoptimized assay format; the sensitivity of melittin as representative biological pore of similar charge and aggregation number to detect the same reaction is more than four orders of magnitude inferior

Synthetic Catalytic Pores

Catalytic activity of a synthetic multifunctional pore is studied in large unilamellar vesicles under conditions where substrate and synthetic catalytic pore (SCP) approach the membrane either from the same side (cis catalysis) or from opposite sides (trans catalysis). A synthetic supramolecular rigid-rod ß-barrel with excellent ion channel characteristics is identified as SCP using 8-acetoxypyrene-1,3,6-trisulfonate (AcPTS) as model substrate. The key finding is that application of supportive membrane potentials increases the initial velocity of AcPTS esterolysis (v0). This results in an increase of Vmax beyond experimental error (+30%), whereas KM increases less significantly. Long-range electrostatic steering by the membrane potential, possibly guiding substrates into the transmembrane catalyst and, more importantly, accelerating product release (foff = 1.3) is discussed as one possible explanation of this global reduction of catalyst saturation. Control experiments show, inter alia, that similarly strong changes do not occur with opposing membrane potentials

Sugar Sensing with Synthetic Multifunctional Pores

Recently, synthetic multifunctional pores have been identified as “universal” detectors of chemical reactions. In this report, we show that with the assistance of enzymes as variable co-sensors, synthetic multifunctional pores can serve as similar universal sensors of variable components in mixed analytes. Sugar sensing in soft drinks is used to exemplify this new concept. This is achieved using invertase and hexokinase as co-sensors and a new synthetic multifunctional pore capable of discriminating between ATP and ADP in an “on-off” manner as sensor. The on-off discrimination between ATP as good and ADP as poor pore blocker is shown to be reasonably tolerant of changing experimental conditions. These results identify universal sensing with synthetic multifunctional pores as a robust, sensitive, and noninvasive method with appreciable promise for practical applications

Blocker Efflux Through Blocked Pores

The objective of this paper is to clarify a frequent confusion that occurs with synthetic multifunctional pores: does molecular recognition by a synthetic pore exclude molecular translocation through the same pore? Evidence that this is not the case is provided with a set of competitive experiments using a rigid-rod ß-barrel with internal histidine-arginine dyads as synthetic multifunctional pores (SMPs) as well as 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) as a fluorescent and 1,3,6,8-pyrenetetrasulfonate (PTS) as a non-fluorescent blocker. Direct evidence for the efflux of HPTS blockers through blocked SMPs is obtained by quenching of released HPTS with an externally added quencher. Blockage of blocker efflux through blocked pores was demonstrated by adding PTS as the opposing external blocker (KD = 3.3 µM). A Hill coefficient of n = 1.5 may indicate that binding of more than one PTS blocker is necessary to inhibit the efflux of HPTS blockers. Supported by structural information on blockage and selectivity from biological potassium channels, blocker efflux through blocked pores is discussed as being implicated in selectivity

Outer Surface Modification of Synthetic Multifunctional Pores

The characteristics of pores formed by p-octiphenyl beta-barrels with LWV triads at the outer surface are reported in comparison with the conventional rigid-rod beta-barrels with all-L outer surface. Maintained multifunctionality of tetrameric pores with external LWV triads (inversion of ion selectivity, molecular recognition and transformation) is implicative for intact barrel interior. Increased pore activity supports dominance of high bilayer affinity for W over low affinity for V. Transmembrane p-octiphenyl orientation (from fluorescence depth quenching) supports barrel-stave (rather than toroidal) pores and dominance of transmembrane preference of rigid rods over interfacial preference of W. Destabilization of beta-barrel pores in membranes (from short single-channel lifetimes) and in the media (from 4th-power dependence on monomer concentration) by LWV triads supports dominance of low beta-propensity for W over high beta-propensity for V. The relation between the stability of supramolecular (pre)pores and dependence of activity on monomer concentration is discussed in a more general context

Synthetic multifunctional pores: deletion and inversion of anion/cation selectivity using pM and pH

We report the characterization of multifunctional rigid-rod [beta]-barrel ion channels with either internal aspartates or arginine–histidine dyads by planar bilayer conductance experiments. Barrels with internal aspartates form cation selective, large, unstable and ohmic barrel-stave (rather than toroidal) pores; addition of magnesium cations nearly deletes cation selectivity and increases single-channel stability. Barrels with internal arginine–histidine dyads form cation selective (P[K+]/P[Cl–]= 2.1), small and ohmic ion channels with superb stability (single-channel lifetime > 20 seconds). Addition of "protons" results in inversion of anion/cation selectivity (P[Cl–]/P[K+]= 3.8); addition of an anionic guest (HPTS) results in the blockage of anion selective but not cation selective channels. These results suggest that specific, internal counterion immobilization, here magnesium (but not sodium or potassium) cations by internal aspartates and inorganic phosphates by internal arginines (but not histidines), provides access to synthetic multifunctional pores with attractive properties

Bioorganic Chemistry of Rigid-Rod Molecules: Adventures with p-Oligophenyls

Studies on the usefulness of rigid-rod molecules to address pertinent questions of biological relevance are summarized. Emphasis is placed on (a) the supramolecular functional plasticity of p-octiphenyl ß-barrels expressed in molecular recognition (adaptable synthetic hosts), molecular translocation (adaptable synthetic ion channels) and molecular transformation (esterases, RNases), (b) molecular recognition of polarized membranes by rigid push-pull rods, as well as (c) the synthetic organic chemistry of rigid-rod molecules